Our major goal is to determine the functions of macroH2A histone variants at the molecular, cellular and organismal levels. MacroH2As have an unusual structure consisting of a full-length H2A-region linked to a large C-terminal nonhistone-region that comprises more than half of the protein. MacroH2As are highly conserved among vertebrates indicating that they have important function(s), and in mammals are highly concentrated in the inactive X chromosome suggesting a role in gene regulation. The involvement of macroH2A proteins in processes other than X inactivation is indicated by their presence at many other locations in the chromatin of both female and male cells and by their high evolutionary conservation in non- mammalian vertebrates that do not have X inactivation. There are two macroH2A genes in mice and humans, macroH2A1 and macroH2A2. In order to identify the specific functions of macroH2A variants, we have produced macroH2A knockout mice and are identifying genes and repetitive elements that have altered expression in macroH2A knockout mouse tissues and cells. We have developed a novel method of purifying macroH2A nucleosomes and will use DNA from these nucleosomes to examine the distribution of macroH2A nucleosomes in the chromatin. Two major goals of these studies are to identify likely sites of macroH2A action and to gain insights into the mechanisms that localize macroH2A to specific regions of chromatin. We will investigate the mechanisms of macroH2A function by determining the effect of macroH2A knockouts on the structure of the chromatin at target sites identified by our other studies. We are also examining these mice to identify specific physiological effects produced by the absence of macroH2A proteins. Our studies of macroH2A1 knockout mice indicate a role for these proteins in regulating lipid and glucose metabolism. PUBLIC HEALTH RELEVANCE Metabolic disorders such as diabetes and obesity are major human health issues. Better understanding of normal mechanisms of metabolic regulation may provide new approaches for the diagnosis and treatment of these and other metabolic diseases.